Summary The nucleoid structuring protein H-NS plays important roles in the transcriptional silencing of many bacterial genes. Proteins that alleviate this silencing are central to bacterial physiology and control many processes including virulence gene expression, biofilm formation and bacterial adaptation to stress. Despite recent advances, transcriptional silencing and anti-silencing mechanisms remain poorly understood. An improved understanding of these processes could lead to the discovery of novel drug targets and/or development of new antibacterials. Here we propose to use the bacterial pathogen Shigella as a model system to study this mechanism of transcriptional regulation. The icsP gene, which is carried by the Shigella virulence plasmid, is transcriptionally repressed by H-NS and derepressed by the transcription factor VirB. Two sites (essential sites), located over 1 kb upstream of the icsP transcription start site, match the reported VirB binding sites and are required for transcriptiona anti-silencing. Our long term goal is to understand how VirB alleviates H-NS- mediated repression of virulence genes in Shigella flexneri. The overarching hypothesis of this project is H- NS-mediated transcriptional repression of the Shigella icsP promoter is relieved when VirB binds to the essential sites, oligomerizes along DNA and triggers localized changes in DNA supercoiling. Our Specific Aims are: 1) to determine the role that the essential sites play in Vir oligomerization along DNA, 2) to determine the role that VirB-induced changes in plasmid supercoiling play in transcriptional anti-silencing by VirB, and 3) to determine whether the essential sites are sufficient for VirB binding in vivo; a key step in VirB- dependent transcriptional anti-silencing. The work we propose challenges classical views of the system we study, and a new genetic tool will be created to allow VirB binding to be assessed in vivo. Our project can therefore be considered conceptually and methodologically innovative. All experiments proposed in this application have been carefully selected so they are suitable for the graduate and undergraduate students at the University of Nevada, Las Vegas. By testing our overarching hypothesis we will learn more about the VirB protein, which can be considered the molecular switch that controls Shigella virulence, its interplay with the nucleoid structurin protein H-NS and the mechanisms that allow transcriptionally silenced genes to become active in Shigella and possibly other bacteria.